In the past year, the FDA, the EMA or the MHRA authorised fifty-three (53) novel drugs. While this 2024 harvest is not as rich as that of 2023, when 70 new chemical entities were approved, the number of “orphan” drug authorisations in 2024 (21) is similar to that of 2023 (24), illustrating the very dynamic development of therapeutics in areas of high unmet need. Clearly, the most striking characteristic of the 2024 drug yield is the creative pharmacological design, which allows these medicines to employ a novel approach to target a disease. Some such 2024 notable examples are: the first drug successfully using a ”dock-and-block” mechanism of inhibition (zenocutuzumab), the first approved drug for schizophrenia designed as an agonist of M1/M4 muscarinic receptors (xanomeline), the first biparatopic antibody (zanidatamab), binding two distinct epitopes of the same molecule, the first haemophilia therapy that instead of relying on external supplementation of clotting factors, restores Factor Xa activity by inhibiting TFPI (marstacimab), or the first ever authorized direct telomerase inhibitor (imetelstat) that reprogrammes the tumour cells’ oncogenic drive. In addition, in 2024 an impressive percentage of the novel drugs were first-in-class (28 out of 53, or 53% of the total) and a substantial number of them can be considered disease-agnostic, indicating the possibility of future approved extension of their use into additional indications. Overall, the 2024 harvest demonstrates the therapeutic potential of innovative pharmacological design, which allows the effective targeting of intractable disorders and addresses crucial, unmet therapeutic needs

Background and Purpose. Heroin and cocaine users tailor their dosage, frequency, and method of administration, to maximize the drugs’ effects or prevent withdrawal symptoms. Counterintuitively, preclinical self-administration and choice experiments employ fixed unit-doses and timeouts (after doses) largely resulting in uniform drug-taking patterns. The application of these procedures also disregards the distinct pharmacokinetic properties of heroin and cocaine. This uniformity contrasts with the significantly different ways humans tailor their dosage and frequency of heroin and cocaine use. Here, by combining behavioral and pharmacokinetics assessments we revealed that self-administration procedures lacking the timeout may overcomes this limitation. Experimental Approach. We analyzed heroin and cocaine taking- and seeking-patterns and estimated drug-brain levels in the presence or absence of timeout. We further assessed how absence of timeout and the availability of drug or social peer (access time to the two rewards) affect drug preference in choice procedures. Key Results. Removing the timeout had a profound effect on heroin-taking patterns and seeking, promoting the emergence of burst-like intake, yielding higher brain peak concentrations of heroin. Timeout removal had marginal impact on cocaine taking patterns and seeking. Increasing the access time to the drug in the discrete choice procedure resulted in higher drug intake and increased preference for heroin, but it did not alter cocaine preference. Conclusion and Implications. Removing timeout during self-administration revealed distinct heroin and cocaine taking patterns. Self-administration without timeout and access to high heroin doses during choice more closely mimic human heroin taking patterns and related behaviors, including maladaptive choices.

Background and Purpose: Platelet function during inflammation is dependent on activation by endogenous nucleotides acting on purinergic receptors. The P2Y14 receptor (P2Y14R) has been reported to be expressed on platelets and is involved in leukocyte recruitment during inflammation. However, a role for P2Y14R receptors on platelet function has not yet been determined. Experimental Approach: Platelets obtained from healthy human volunteers were incubated with the P2Y14R agonist, UDP-Glucose (UDP-G), and PPTN, a selective P2Y14R antagonist. Platelet activation was quantified using Ca2+ mobilization, aggregation, and chemotaxis assays. Cooperativity with P2Y1 receptor (P2Y1R) activation was also assessed after stimulation with UDP-G in the presence of MRS2500, a selective P2Y1R antagonist. Key Results: Ca2+ mobilization occurred in platelets after incubation with UDP-G in a concentration-dependent manner, and this was suppressed in platelets treated with PPTN. Platelets did not aggregate, or bind to fibrinogen after incubation with UDP-G. However, platelet chemotaxis towards f-MLP was dependent on P2Y14R stimulation with UDP-G and this was reduced by Rho-GTPase inhibitors. Furthermore, UDP-G induced Ca2+ mobilization and chemotaxis were also inhibited when platelets were pretreated with MRS2500. Conversely, ADP induced Ca2+ mobilization, chemotaxis and aggregation were not affected by the incubation with PPTN. Conclusion and Implications: Platelets can be activated via P2Y14R stimulation to induce chemotaxis but not aggregation. Furthermore, this was dependent on concomitant activation of P2Y1R. Activation of P2Y14Rs on platelets may therefore be relevant during inflammation, but cooperation with P2Y1R activation is required.

Background and Purpose: Preclinical studies suggest that highly selective dopamine D3 receptor (D3R) antagonists or partial agonists hold promise for treating substance use disorders (SUD). However, their limited effectiveness in reducing cocaine self-administration is a major drawback. This study investigated whether cariprazine (a D3R-preferring partial agonist) and its analogs ESG-1-60 and ESG-1-61 have enhanced efficacy in reducing cocaine-taking and -seeking behavior. Experimental Approach: In vitro BRET experiments were used to characterize the functional efficacies of cariprazine and its analogs. Intravenous cocaine self-administration and reinstatement models were used to evaluate efficacy in reducing cocaine-taking and -seeking behavior. Optical intracranial self-stimulation (oICSS) procedures assessed effects on DA-dependent behavior. Open-field locomotion, oral sucrose self-administration, and conditioned place-preference were used to evaluate potential unwanted side effects. Key Results: BRET functional assays indicated that cariprazine and ESG-1-60 are D3R-preferring partial agonists, while ESG-1-61 is a D3R-preferring antagonist/inverse agonist. All three compounds inhibited cocaine self-administration under both fixed-ratio and progressive-ratio reinforcement schedules and reduced cocaine-induced reinstatement of drug-seeking behavior in both male and female rats. The compounds did not alter locomotor behavior but suppressed sucrose intake and DA-dependent oICSS. Cariprazine and ESG-1-61 produced significant place aversion, while ESG-1-60 did not. Chronic administration of ESG-1-60 inhibited cocaine self-administration. Conclusions and Implications: Novel D3R-preferring compounds ESG-1-60 and ESG-1-61 are highly effective in reducing cocaine taking and seeking under various reinforcement conditions. ESG-1-60 warrants further investigation as a new pharmacotherapy for treating cocaine use disorder due to its effectiveness in these models and lack of unwanted behavioral effects.

Drug development for children presents unique challenges and is highly regulated. Novel approaches, such as the use of extrapolation to address for example the need to avoid unethical studies, whilst supporting robust evidence generation have been developed in support of benefit/risk considerations by regulatory authorities. This is only one step in the decision-making process towards access, which in Europe also includes health technology assessment (HTA) bodies. Discussions related to evidentiary requirements in small populations through the use of evidence transfer has been identified as a priority action by EMA/ EUnetHTA 21. We describe the outcome of this work; reflect on the discussions taken place how to leverage prior knowledge through identifying and addressing uncertainties during life cycle management to support regulatory and HTA decision making. Through examples, we discussed the spectrum of use to support evidence generation, and developed regulatory and HTA reflections on general design considerations important for robust evidence generation; reflective of the joint ambition. Early interactions with all respective stakeholders, particularly between regulators and HTA bodies are key to optimise data generation and utility in children. In Europe, the HTA regulation will offer opportunities for collaborations, which are important for all development efforts. We collaboratively explored the unique specific challenges relating to paediatric drug development, ethically and in its ability to leverage prior knowledge, as exemplified using extrapolation. Learnings from these offers opportunities to further develop methodology how to leverage uncertainties across a product’s life cycle for small populations generally.

Background and Purpose: Pharmacokinetics have traditionally been assessed using concentration measurements from methods with low temporal resolution, such as blood draws, leading to profiles being estimated from sparse or blended data. Recent advances in in vivo sensors, however, now enable the collection of hundreds of observations over a few-hours for each individual drug administration. Previous analyses of such data for the antibiotic tobramycin have identified significant (several-fold), hours-scale changes in the efficiency with which this renally cleared drug is eliminated. Here we apply similar analyses to study the pharmacokinetics of another renally cleared drug, the antibiotic vancomycin. Experimental Approach: We estimate vancomycin pharmacokinetic profiles using previously collected time-dense plasma concentration measurements within six anesthetized rats. Specifically, we fit standard one- and two-compartment models, as well as time-varying one-compartment models (in which the proportionality relating concentration to elimination rate is time-varying), to these data to investigate if the time-varying models are statistically preferred for describing individual-level vancomycin pharmacokinetics, over standard one- and two-compartment models. Key Results: One-compartment models incorporating time-varying elimination proportionalities are statistically preferred over standard one- and two- compartment models for 5 of our 6 vancomycin time courses. When the initial impact of the distribution phase is removed from these data, a reciprocally time-varying one-compartment model is preferred over the standard-one compartment model in 4 of 5 considered datasets. Conclusion and Implications: These results provide further animal-model evidence that the pharmacokinetics of renally cleared drugs can vary significantly over timescales as short as a few hours.

Background and purpose Diabetic nephropathy (DN) is a leading cause of chronic kidney diseases (CKD) characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signaling in diabetic kidney fibrosis to reveal potential therapeutic target. Experimental Approach Db/db mice were used to study DN. RNA sequence analyses, western blot and immunostaining were conducted to profile gene expression in kidneys. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signaling microdomains in MCs. Rolipram, a PDE4 inhibitor, was orally administrated in db/db mice to test its impact on the kidney. Key Results Db/db mice exhibited impaired kidney function with elevated adrenergic and fibrotic gene expression, including ADRB1, PDEs, ACTA2, and TGF-β. MCs with dysregulated YAP pathway played a pivotal role in extracellular matrix secretion in DN. TGF-β up-regulated β1-adrenergic receptors (β1ARs) and α-SMA in MCs. Diminished nuclear-specific cAMP signaling in MCs by TGF-β triggered profibrotic gene transcription via reducing PKA-dependent phosphorylation of YAP. The profibrotic formation was partially alleviated by norepinephrine administration and prevented by combined treatment of norepinephrine and rolipram. In db/db mice, rolipram treatment alleviated kidney fibrosis and restored kidney filtration function. Conclusion and Implications DN impairs nuclear-localized β1AR-cAMP signaling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in DN.

Background: Previous research demonstrated that emodin inhibits cardiac fibrosis through metastasisasspcoated protein 3 (MTA3), but its limited bioavailability hinders clinical application. Aim: To enhance emodin clinical potential, a new derivative, emodin succinyl ethyl ester, was synthesized by modifying the 3’-OH position. This study assessed its drug-likeness, anti-fibrotic properties, and molecular mechanisms involving MTA3. Methods: Drug-likeness properties of the emodin derivative were evaluated using computational-aided drug design (CADD) approaches. An animal model of transverse aortic constriction (TAC)-induced cardiac fibrosis and Angiotensin II (AngII) stimulated cardiac fibroblasts were used in vivo and ex vivo, respectively, to determine the effects of emodin derivative on cardiac fibrosis and fibroblast transdifferentiation. Bioinformatics analysis of the PROMO database, CADD, chromatin immunoprecipitation (ChIP), luciferase reporter assays, and functional experiments were employed to predict, identify and validate the relationship between MTA3 and its upstream transcription factors. Results: Emodin derivative exhibited superior drug-likeness and anti-fibrotic effects compared to emodin by effectively inhibiting cardiac fibroblast transdifferentiation and restored MTA3 expression. E2F1 was identified and validated as a transcriptional regulator, promoting α-SMA and COL1A2 expression, and directly reducing MTA3 expression in cardiac fibroblasts. The emodin derivative demonstrated stronger binding to E2F1 transcription site than emodin, reducing E2F1 expression and enhancing anti-fibrotic action via MTA3. Conclusion: The emodin derivative shows improved drug-likeness and potent inhibition of cardiac fibrosis by targeting E2F1, disrupting its pro-fibrotic function, restoring MTA3 expression, and halting fibrosis progression. This advances emodin’s potential as a clinical therapy for cardiac fibrosis and provides insights into its anti-fibrotic mechanisms.

Background and purpose Currently, the clinical benefits of liver cancer treatment are still limited. Emerging evidences have highlighted that paraptosis may be an effective strategy to threapy liver cancer. In our previous studies, compound 4a was found to induce paraptosis in cancer cells. Here, the characteristics of compound 4a induced paraptosis were further revealed, and for the first time, the target and related molecular mechanisms of compound 4a induced paraptosis in liver cancer were explored. Study design and methods The effect and mechanism of compound 4a on liver cancer cells both in vitro and in vivo. And the targets of compound 4a that triggering paraptosis, were identified and confirmed by using the technology of mass spectrometry-based drug affinity responsive target stability(DARTS), siRNA, and Cellular thermal Shift Assay(CETSA). Intracellular calcium concentration and protein distribution were detected by Cal520-AM and immunofluorescence, respectively. Results We found that compound 4a can effectively induce paraptosis-like cell death in liver cancer both in vitro and in vivo, and its effect is comparable to the first-line anti-liver cancer drugs oxaliplatin while with higher safety. We identified that Calreticulin(CRT) protein as a target of compound 4a, which causing cellular endoplasmic reticulum(ER) stress and calcium overload. CRT knockdown increased cell proliferation and reduced cytoplasmic vacuoles, which was associated with inhibited ER stress and paraptosis. Conclusions Our study provides a potential safe and effective agent for the treatment of liver cancer. Moreover, we have clarified characteristics of compound 4a-triggered paraptosis and revealed a unique function of CRT in paraptosis.

Background and Purpose Emerging evidence has suggested that somatic stimulation, including electroacupuncture (EA), could produce autonomic reflexes to modulate visceral functions. However, the efficacy and underlying mechanisms for EA on allergic pulmonary inflammation (API) remain elusive. Experimental Approach Papain-induced API mice were treated with distinct current-intensity EA at the back BL13, hindlimb ST36, and forelimb LU5 acupoints. The change in API was determined by whole-body plethysmograph (WBP), immunohistochemistry, flow cytometry, and HE staining. We employed lung functional testing combined with pharmacological inhibition and optogenetic activation approaches to examine the underlying mechanisms for EA effects on API. Key Results EA at the back BL13 region, but not limb regions, via a current-intensity-dependent manner, exacerbated the API. Pharmacological blocking of the local thoracic sensory or the lung-innervated autonomic nerve eliminates the EA-produced detrimental effects. Chemical pulmonary sympathectomy was further enhanced, but inhibition of the upregulated muscarinic M3 acetylcholine receptor within the pulmonary was sufficient to protect from the inflammation-exacerbation effect of EA. Conclusions and Implications Our findings suggest that BL13 EA evokes a somatic-autonomic-pulmonary M3 pathway to enhance API. The revelation of somatotopic organization and intensity-dependency in driving the pulmonary autonomic pathways could help optimize stimulation parameters to improve both efficacy and safety in various forms of physical therapy, including bioelectronic medicine and traditional as well as modern forms of moxibustion and EA, to modulate API.

This brief review highlights some of the structure-activity relationships of classic serotonergic psychedelics. In particular, we discuss structural features of three chemotypes: phenethylamines, ergolines, and certain tryptamines, which possess psychedelic activity in humans. Where it is known, we point out the underlying molecular mechanisms utilized by each of the three chemotypes of psychedelic molecules. With a focus on the serotonin 5-HT2A receptor subtype, a G-protein coupled receptor known to be the primary target of psychedelics, we reference several x-ray and cryoEM structures with various ligands bound to illustrate the underlying atomistic basis for some of the known pharmacological observations of psychedelic drug actions.

Mechanopharmacology is an emerging interdisciplinary field that investigates drug action using biomechanically appropriate in vitro systems to the relevant (patho)physiology. This review outlines emerging technologies and techniques which aim to bridge the gap between mechanical cues influencing cellular biology and conventional pharmacology. We delve into the impact of mechanopharmacology on drug development in cancers and fibrotic diseases. Mechanical cues such as stretch, stiffness, circadian rhythms, fluid flow, intercellular signalling cascades and cytoskeletal structures can modulate drug interactions with molecular targets with implications for drug discovery and development. Models incorporating mechanopharmacological cues to investigate pharmacokinetics, pharmacodynamics and therapeutic outcomes are outlined. Furthermore, this review discusses innovations in the use of biomaterials and microfluidics, to enable further the emulation of the mechanical microenvironment. We advocate for the application of mechanopharmacological considerations to improve the physiological relevance of methods used in the drug discovery pipeline.

Background and purpose: Several analgosedative drugs used in the management of critically ill patients impair gastrointestinal (GI) propulsion and thereby carry a risk for developing sepsis. The gut microbiota has emerged as a factor that can influence GI motility, but whether GI microbial disruption modifies GI peristalsis impairment by analgosedative drugs has not yet been analysed. This question was addressed in the guinea-pig small intestine following antibiotic-induced depletion of the GI microbiome. Experimental approach: Guinea-pigs were enorally pretreated with meropenem, neomycin and vancomycin, and antibiotic-induced depletion of the GI microbiome was confirmed by 16S rDNA sequencing. Peristalsis in the isolated guinea-pig small intestine was evaluated by assessing the peristaltic pressure threshold at which a peristaltic wave is triggered. The expression of factors that may be relevant to communication between GI microbiota and motor system was examined at the mRNA (qPCR) and/or protein (ELISA) level. Key results: Antibiotic treatment disturbed the small intestinal microbiome as shown by a decrease of bacterial load and α-diversity. Microbial disruption did not affect peristalsis at baseline but blunted the ability of α2-adrenoceptor (ADRA2) agonists to inhibit peristalsis, while the anti-peristaltic effects of sufentanil, midazolam, neostigmine and propofol were inconsistently affected. These functional alterations were complemented by a decreased expression of ADRA2, TLR3, TLR4 and TLR7, IFN-γ and NOS2. Conclusions and implications: Antibiotic-induced disturbance of the GI microbiome selectively blunts the ability of ADRA2 agonists to impair peristalsis. This effect is explained by decreased ADRA2 expression, which may arise from TLR downregulation in the dysbiotic gut.

Background and Purpose: Given the recent rise in cannabis legalization, accessibility and consumption by pregnant individuals, there are unintended developmental consequences considering the key role that the endocannabinoid system plays in fetal development and later-life energy homeostasis. Whether perinatal cannabis exposure (PCE) affects energy homeostasis positively or adversely in adulthood is unknown. We explored the long-term effects of maternal voluntary oral cannabis consumption on the metabolic outcomes of high fat diet (HFD) in adult offspring. Experimental Approach: Pregnant mice voluntarily consumed cannabis from gestational day 1.5 until postnatal day (PD) 10. Pregnancy and pup outcomes and active maternal behavior were recorded. Male and female offspring (PD49) were placed on a 12-week HFD or control diet; their weight gain, adiposity, glucose tolerance, insulin sensitivity, circulating hormones, and pancreas structure were measured. Key Results: PCE pup weight was reduced but was restored by PD16. PCE did not influence weight gain or metabolic characteristics of male mice on a HFD. However, PCE female offspring on a HFD had reduced accumulation of adipose tissue and lower insulin, leptin, and resistin independent of body weight and while PCE females on control diet showed altered basal insulin sensitivity likely due to increased glucagon levels in parallel with reduced islets of Langerhans size and enhanced gene expression of cannabinoid 2 receptors in white adipose tissue. Conclusion and Implications: PCE influences metabolic outcomes in female offspring; it adversely affected glycemic control in female offspring on control diet while it mitigated HFD-induced metabolic dysfunction. This raises concerns about the long-term effects of PCE on the metabolic health of offspring.

Background and Purpose: Hyoscine butylbromide (HBB) has a low oral (PO) bioavailability. Further, limited data on its activity on non-gastrointestinal (GI) smooth muscle spasms after oral dosing are available, meaning its effect outside the GI tract has been questioned. This pharmacokinetic/pharmacodynamic (PK/PD) study, conducted using female rats, aims to cover this gap. Experimental Approach: PK study: HBB and atropine (comparator) were administered PO and IV to rats, and concentrations in plasma, and colon, uterus and urinary bladder (CUB) were measured. PD study-1: Concentration–response (C-R) curves of HBB and atropine (10-9–10-4M) were obtained for carbachol (CCH)-induced (10-5 M) pre-contracted CUB; PD study-2: CUB were pre-incubated with HBB and atropine at maximum concentrations (Cmax) from PK study, and CCH C-R curves (10-9–10-4 M) were obtained; PD study-3: HBB and atropine were administered PO and IV to rats as for PK study, CUB were collected at 0.5 h (IV) and 4 h (PO), and CCH C-R curves (10-9–10-4 M) were obtained. Key Results: PO HBB showed higher Cmax in CUB (192.5, 3.70 and 1.85 ng∙g-1, respectively) than plasma (0.008 ng∙mL-1). HBB and atropine reduced (concentration dependently) CCH-induced contractions in CUB. PO HBB showed highest spasmolytic activity in colon (40%), followed by uterus (30%) and urinary bladder (10%). Conclusion and Implications: This is the first comparative study investigating PO and IV HBB and atropine in GI and non-GI tissues. Despite low bioavailability, PO HBB accumulates and exerts spasmolytic effects outside the GI tract.

Background and Purpose: Fentanyls and nitazenes are μ opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO. Experimental Approach: In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Key Results: The rate of agonist dissociation from the μ receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and Implications: With fentanyls and nitazenes, that slowly dissociate from the opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

Background and Purpose: Fibroblast Growth Factor (FGF), VEGFR2, and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG35-55-induced EAE prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSF1R and VEGFR2 by AZD4547 may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model. Experimental Approach: Female C57BL/6J mice were treated with AZD4547 (6.25 mg kg-1 or 12.5 mg kg-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (day 18/20 p.i.) and the chronic phase of EAE (day 41/42 p.i.). Key Results: In the prevention experiment, treatment with AZD4547 prevented severe first clinical episodes by 66.7 or 84.6% respectively. Mice treated with 12.5 mg kg-1 of AZD4547 hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, treatment with AZD4547 resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased in both experimental approaches. Conclusion and Implications: Multi-kinase inhibition by AZD4547 in a well-tolerated dose of 1 mg kg-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

The total iridoid glycoside extract of Lamiophlomis rotata (IGLR) has been proven to be the main pharmacological ingredient for wound healing; however, it is still unclear, which compounds in the extract are responsible for this effect. We developed and validated a mass spectrometry imaging (MSI) method to reveal the relationship between phytochemical components and the pharmacological efficacy in IGLR. Shanzhiside methyl (SM) ester, 8-O-acetylshanzhiside methyl ester (ASM), and Phlorigidoside C (PhC) have been identified as quality markers in IGLR to accelerate wound healing by MSI method. As per MSI data with UMAP manifold analysis, these compounds overlapped in the same class with endogenous metabolites (lactate, citrate and succinate). In addition, UPLC-Q/TOF-MSn coupled with multivariate analyses for skin tissues in the normal and wound groups further confirmed the results. Furthermore, MSI data from livers and kidneys revealed that SM, ASM, and PhC absorption significantly increased, whereas their elimination rates remarkably decreased in the injured group. Collectively, the overall data demonstrated that SM, ASM, and PhC selectively increased in the new granulation tissue and that the bioavailability of these three quality markers was remarkably improved in the wound group after continuous gavage for 7 days. This protocol can be applied to reveal the relationship between phytochemical components and the pharmacological efficacy of other iridoid glycoside extracts.